23. Errors I. Proofreading and MMR (1,402; 7/30)

DNA polymerases are astonishingly accurate--but not perfect. Let me quantify that.

The leading and lagging strand DNA polymerases, which are the workhorses of genome replication, initially make a nucleotide addition mistake once every 10,000 to 100,000 nucleotides. That’s impressive, but not nearly accurate enough. That number of mis-incorporations would result in tens to hundreds of thousands of errors in a replicated human genome.

But it's known that when a human genome is replicated, it contains very few errors--zero to a handful. So something else must be going on. That "something else" is DNA repair.

The two repair solutions we're about to discuss, DNA polymerase proofreading and a multi-protein pathway called mismatch repair (MMR) reduce the number of errors to close to zero. Proofreading is performed by the polymerase itself when it senses an error. Proofreading reduces errors by roughly 100- to 1,000-fold. MMR delivers further significant improvement that can also reach hundreds- to roughly a thousand fold.

These two repair solutions brings the number of errors in a replicated genome down to what scientists have observed: a very small number of errors, or even no errors. So, once again, the cell's got this covered!

You might ask why we're devoting time to DNA repair in a book about DNA replication? Because the two are inseparable. Replication inevitably generates errors. Those errors must be fixed. Replication and repair are so tightly linked that we can't ignore the topic. And we wouldn’t want to. DNA repair is among the most remarkable abilities of cells.

Types of Errors

Before describing these two error correction solutions, let me clarify what I mean by an error. For the moment, we'll consider three kinds of errors in the strand being synthesized: substitutions, insertions, and deletions

Substitutions create nucleotide mismatches. The DNA polymerase simply adds the wrong nucleotide. It inserts, for example, an "A" nucleotide across from a parental template strand "C" instead of the correct "G". Substitutions are like misspellings.

The other two kinds of errors are insertions and deletions. An insertion is the addition of an extra, unpaired nucleotide on the new strand being synthesized. A deletion is the failure to add a paired nucleotide--one that ordinarily should be there--on the new strand.

Here's an example that's closer to home. A letter "t" insertion in the word "nucleotide" might result in the misspelled word "nucleottide" with an extra unwanted "t." A letter "t" deletion might result in the misspelling "nucleoide" with no "t."

Insertions and deletions often occur when a DNA polymerase slips when copying a repetitive sequence--a long stretch of As, for example. The enzyme in a sense loses track of where it is on the parental template strand.

With an insertion, the extra nucleotide on the new strand doesn't pair with any nucleotide on the template strand. So it generates a small loop or protrusion on the new strand. With a deletion error, the unpaired nucleotide on the parental template strand forms the loop. So both scenarios result in small loops, but on opposite strands.

DNA polymerase "proofreading"

The first line of defense against errors is DNA polymerase proofreading. Both the leading and lagging strand DNA polymerases possess a second enzymatic activity in addition to their primary DNA polymerization activity. It is referred to as a "3′→5′ exonuclease activity." 3' to 5' is the direction opposite that of DNA synthesis. This enables the enzyme to quickly chew back the DNA strand it's synthesizing to immediately remove the error.

Here's how it works. When there's a mismatch or an insertion-deletion loop near the growing 3' end of the new strand, it distorts the geometry of the double helix and slows the polymerase. The enzyme senses this and moves the 3' end of the new strand from the polymerase active site to the exonuclease active site.

The enzyme then literally steps backward and chews off the incorrect nucleotide. Once the error is removed, the 3' end of the new strand shifts back to the enzyme's main polymerization active site and synthesis resumes.

DNA polymerase proofreading is like the "delete" key on your keyboard. When you make a typo, you hit delete to remove it. Then you keep typing. This is a pretty good analogy for DNA polymerase proofreading.

As I mentioned, DNA polymerase proofreading improves fidelity by roughly 100- to 1,000-fold. But this would still leave more errors than the cell can safely tolerate. Thus, human cells employ another error-fixing solution: MMR.

Mismatch repair (MMR)

Unlike proofreading, MMR involves multiple steps and multiple enzymes. And whereas proofreading occurs immediately by the polymerase itself. MMR acts soon afterward.

The pathway initiates with detection of the error at or near the replisome. But it often finishes behind the moving replication fork after the polymerase has moved beyond the error.

At this point, I'd like to step back and generalize, because MMR and most of the other repair pathways I'll introduce shortly follow a common logic known as “cut-and-patch repair."

Here's a high-level overview of this process. In cut-and-patch repair, a specific protein recognizes the error and then another--an endonuclease--cuts that strand in front of the error (5' of the error). Next, an exonuclease starts at that cut and chews up the DNA to remove the error. Then a DNA polymerase and its partner PCNA replace those nucleotides. Finally, a DNA ligase seals the nick left by the polymerase.

Let’s now focus on MMR, specifically.

The primary MMR detector protein complex--and the initiator of the pathway--is the C-shaped MutSα. It specifically recognizes single-base mismatches and small insertion–deletion loops of one or two nucleotides. These represent most replication errors.

MutSα diffuses through the nucleus during S phase when the new genome is being synthesized. But MutSα also contains a PIP-box with which it transiently and repeatedly binds to PCNA. This on-again-off-again binding increases the concentration of MutSα near replication forks--that is, exactly where new errors can be found.

As DNA is synthesized, MutSα repeatedly binds to DNA and probes it for the kinds of abnormalities caused by substitutions, insertions, and deletions. Mismatched base pairs and small insertion-deletion loops make the DNA more flexible than correctly paired DNA. When MutSα encounters one, it grips the DNA and bends, or kinks, it. Then, a specific amino acid in MutSα wedges between the base pair that includes the mismatched base. It's testing whether it's really an error and also stabilizing the kinked state.

Recognition of the mismatch also promotes ATP binding to MutSα, which changes its shape from its mismatch-recognition C-shape to more like a sliding clamp that encircles the DNA. In its ATP-bound form, MutSα loosens its grip on the mismatch and slides down the DNA. It also helps recruit and activate other repair proteins, including the next enzyme in the pathway, an endonuclease.

Think of an endonuclease as like DNA scissors. It cuts DNA at an internal site. In MMR, the endonuclease makes one or more cuts in the newly synthesized strand. One cut must lie on the 5' side of the mismatch to provide an entry point for the next enzyme, a specific exonuclease.

Starting at the endonuclease cut, the exonuclease chews up the DNA in the direction of the error. (Think of Pac-Man in action!) Other repair proteins regulate the exonuclease so that it removes only a limited stretch of the new strand--a stretch that includes the error.

With the error now removed, a DNA polymerase arrives and a new PCNA is loaded by Relication Factor C (RFC)--the sliding clamp loader--right where the exonuclease started chewing. Together, they synthesize new DNA using the parental template strand as a guide, filling the gap. When the polymerase reaches the far end of the gap, the missing DNA will have been replaced. The DNA polymerase and its PCNA then depart.

DNA polymerase can replace the missing nucleotides, but it can't seal the final break in the new strand's backbone. A DNA ligase arrives to do that. It seals the remaining nick, restoring the strand's continuity. The error is now corrected.

Mismatch repair is an amazing capability of the cell. It employs multiple specialized enzymes which must act in a prescribed sequence to detect, remove and then replace the error. In a typical human cell division, MMR might correct tens to a few hundred copying errors that escaped proofreading per genome replication.

In the next chapter, we’ll turn to a different kind of DNA polymerase error: the incorporation of an RNA nucleotide into DNA.